Enzyme-linked immunosorbent assay (ELISA) has been used as a versatile and powerful tool both in basic science and clinical diagnostics since the mid-1960s. This assay makes use of the physical connection established between an antigen-recognizing moiety and device capable of generating a visual signal. The specificity of the assay is mostly determined by the first component while the sensitivity is determined by the second. Traditionally, as the antigen-recognizing moiety in ELISA, monoclonal or polyclonal antibodies are used and enzymes such as alkaline phosphatase or horse radish peroxidase serve as the device generating the visual signal.
Often, conventional ELISA is not sensitive enough and a wide range of labeling and signal-enhancement strategies have been developed in an attempt to increase its sensitivity. A so-called DNA-enhanced immunoassay strategy stands out as the most sensitive one. This strategy employs antigen-recognizing molecules carrying DNA tags and relies on the ability of DNA polymerases to exponentially amplify these tags. Because of this amplification, the sensitivity of the assay becomes 10-10,000-fold higher than the sensitivity of traditional antigen detection methods. Originally, this strategy was described by Sano et al. in 1992 and was called immuno-PCR (IPCR). Later, a number of modifications of this strategy were developed that targeted two major areas: 1) coupling of immunoassay reagents and DNA markers, and 2) assay readout.
While the original Sano's IPCR was modular and utilized a linker protein that allowed the binding of the Fc part of an IgG and subsequent tagging with biotinylated dsDNA, it has been demonstrated that the performance of pre-assembled antibody-DNA conjugates exceeds that of the stepwise assembled complexes in the modular approach. Several approaches have been proposed that allow direct coupling of DNA tags with antigen-recognizing molecules. For example, Guo et al. described a phage display-mediated IPCR assay that makes use of filamentous bacteriophage M13 to expose on its surface single chain variable fragments (scFv) of IgG antibodies. In this assay, entire phage particles were used for binding to the target antigen and simultaneously their DNA served as a tag that was amplified by PCR upon completion of the antigen-binding portion of the reaction.
Among approaches that try to improve assay readout, the approach termed Expression Immunoassay deserves special attention. It takes advantage of a biotinylated DNA tag which encodes the firefly luciferase. In this approach, formation of the immuno-complex is followed by a one-step, cell-free translation expression step, which enables the detection of 3000 DNA molecules or 50 000 antigen molecules, respectively.
While being substantially more sensitive than conventional ELISA, DNA-enhanced immunoassays turn out to be significantly more difficult to perform and more expensive than conventional ELISA.